This invention relates to a method for the efficient removal, from peptides (including proteins) or salts thereof which possess an optionally oxidized N-terminal methionine residue or diketone of said methionine residue, of the N-terminal methionine residue or the diketone of said methionine residue, in the presence of acetic acid and sodium formate, formic acid and sodium formate, or formic acid and sodium acetate; and to a method for manufacturing peptides or salts thereof which do not possess an optionally oxidized N-terminal methionine residue or diketone of said methionine residue.
When protein is biosynthesized within a cell, its N-terminal is known to start with methionine, which corresponds to the initiation codon AUG of the mRNA. However, as this methionine is removed by subsequent processing, it is usually no longer present in the completed mature protein molecule.
With advancements in recombinant DNA techniques, it has become possible to produce useful proteins using microorganisms and/or animal cells, for example Escherichia coli. There have been instances wherein protein produced via this type of method was found to retain a residue comprised of the aforementioned methionine. For example, the retention rate of methionine was as high as approximately 100% in human growth hormone expressed in E. coli [Nature, 293, 408 (1981)], and 50% in interferon-xcex1 [J. Interferon Res., 1, 381 (1981)], while in nonglycosylated human interleukin-2 the presence of a molecular species with methionine retention on the amino-terminal (N-terminal methionine residue) (Met-rIL-2) has been noted in addition to the molecular species rIL-2 which, like naturally-occurring human interleukin-2, is initiated with alanine.
In regards to method for removing N-terminal amino acid chemically, Dixon reported in 1964 that DL-alanylglycine reacts with glyoxylic acid, pyridine, or cupric acetate in a transamination that results in the production of pyruvoylglycine [Biochem. J, 92, 661 (1964)]. He further reported that thiosemicarbazide reacts with compounds to result in amide bond cleavage, producing glycine [Biochem. J. 90, 2C (1964)]. He subsequently applied this reaction to Pseudomonas cytochrome c-551, reporting the removal of N-terminal glutamic acid [Biochem. J, 94, 463 (1965)].
It is reasonable to speculate that the proteins of molecular species that do or do not possess N-terminal methionine might differ from each other in terms of their superstructure, biological activity, and/or stability, and further, that the addition of methionine to the N-terminal could bring about an increase in antigenicity. It is therefore reasonable to surmise, from the perspective of industrial utility, that it is of significance to establish a method for removing the N-terminal methionine that corresponds to the initiation codon.
With the objective of resolving this problem, a method has been proposed wherein methionine is removed via digestion by cyanogen bromide (BrCN) [Science, 198, 1056 (1977)]. However, in addition to presupposing the absence of methionine residue within the desired mature protein, this method, which subjects the protein to harsh chemical reactions, is by no means conducive to satisfactory results.
Aside from the method described in Unexamined Kokai Application Heisei 10-72489 (EP-A-812856), chemical methods which remove the N-terminal methionine residue from peptides or proteins which possess an N-terminal methionine residue in a selective and efficient manner regardless of the type of peptide or protein, are not known. This is most likely attributable to the difficulty of identifying a chemical reaction that is capable of removing N-terminal methionine under gentle conditions without altering the final product, i.e. the peptide or protein. In particular, as the removal of excess N-terminal methionine from genetically engineered proteins of relatively large molecular weight, particularly those intended for use as pharmaceutical agents, requires the activity of the protein not to deteriorate upon methionine removal, the reaction usually needs to proceed, without heating, in alkalescent to acescent solutions. As this is extremely restrictive by chemical reaction conditions, the current situation has been such that a favorable set of reaction conditions could not be identified.
The inventors, upon diligent study with the objective of providing, via the exclusive cleavage of the N-terminal methionine residue in peptides (including proteins) manufactured via genetic engineering, a method for the manufacture of peptides with amino acid sequences that mimic naturally occurring sequences, have discovered that it is possible to remove, from peptides which possess a diketone of themethionine residue, the diketone of said methionine residue by having a peptide which possesses an optionally oxidized N-terminal methionine residue, as represented in Formula (I) of Scheme 1 below, undergo transamination with, for example, glyoxylic acid, which is an xcex1-diketone, or cupric sulfate, which is capable of providing transition metal. ions, or pyridine, which is a base (for example, an amine), to yield a peptide which possesses a diketone of the said methionine residue, which, when allowed to react with 3,4 diaminobenzoic acid, which is a base (for example, a diamine), in the presence of acetic acid and sodium formate, formic acid and sodium formate, or formic acid and sodium acetate, followed by hydrolysis, it is possible to remove the diketone of the methionine residue from the peptide which possesses the diketone of the methionine residue in an unexpectedly efficient manner. Hence the inventors, having identified a method of removing, at an unexpectedly high yields, the optionally oxidized N-terminal methionine residue from peptides which possess methionine residue, to obtain, without bringing about a deterioration in activity, peptides which do not possess optionally oxidized N-terminal methionine residue, further pursued their studies to achieve the completion of the present invention.
(Scheme 1) 
[In formula (I), m represents the integer 0 or 2, while X can be any peptide chain possessing either an amino acid residue or at least 2 amino acids, although from a practical standpoint, an example would be a peptide chain that corresponds to the X of a protein manufactured via genetic engineering. In the specification of the present patent, the term xe2x80x9cproteinxe2x80x9d or xe2x80x9cpeptidexe2x80x9d may refer to a peptide comprised of multiple amino acids or, in the case of a protein, may refer to either a nonglycosylated or glycosylated peptide or protein.]
In the specification of the present patent, within Scheme 1 above,
The compound represented by general formula (I) can be referred to as xe2x80x9ca peptide which possesses optionally oxidized N-terminal methionine residuexe2x80x9d or xe2x80x9ca peptide which possesses methionine residuexe2x80x9d;
The partial-structure within general formula (I) that is represented as 
[m within the formula having the same meaning as mentioned above] can be referred to as xe2x80x9coptionally oxidized methionine residue,xe2x80x9d xe2x80x9cmethionine residue,xe2x80x9d or xe2x80x9cmethioninexe2x80x9d;
The compound represented by general formula (II) can be referred to as xe2x80x9ca peptide which possesses the diketone of the optionally oxidized N-terminal methionine residuexe2x80x9d or xe2x80x9ca peptide which possesses the diketone of the methionine residuexe2x80x9d;
The partial structure within general formula (II) that is represented as 
[m within the formula having the same meaning as mentioned above] can be referred to as xe2x80x9cdiketone of the optionally oxidized methionine residuexe2x80x9d or xe2x80x9cdiketone of the methionine residuexe2x80x9d; and
The compound represented by general formula (III) can be referred to as xe2x80x9ca peptide which does not possess optionally oxidized N-terminal methionine residuexe2x80x9d or xe2x80x9ca peptide which does not possess the diketone of the optionally oxidized N-terminal methionine residue.xe2x80x9d
Hence, the present invention relates to
(1) a method for removing the diketone of N-terminal methionine residue characterized by having a peptide or the salt thereof that possesses the diketone of an optionally oxidized N-terminal methionine residue, react with 3,4-diaminobenzoic acid or the salt thereof in the presence of acetic acid and sodium formate, or formic acid and sodium formate, or formic acid and sodium acetate,
(2) the method described in (1) above wherein the peptide or the salt thereof which possesses the diketone of the optionally oxidized N-terminal methionine residue is a peptides or the salt thereof that is obtained by having a peptide or the salt thereof that possesses optionally oxidized N-terminal methionine residue react with an xcex1-diketone,
(3) the method described in (2) above wherein the peptide that possesses optionally oxidized N-terminal methionine is a peptide that has been manufactured via genetic engineering,
(4) the method described in (1) above wherein the peptide is a (i) growth hormone, (ii) xcex2-cellulin, (iii) interleukin-2, (iv) neutrophin-3, or (v) apelin,
(5) the method described in (1) above wherein the peptide is a growth hormone,
(6) the method described in (1) above which is characterized by the acetic acid and sodium formate, or formic acid and sodium formate, or formic acid and sodium acetate, being used as a buffering solution of approximately 0.1 to 8 mol/L with a pH of approximately 2 to 9,
(7) a method for removing the diketone of the N-terminal methionine residue characterized by having a peptide or the salt thereof which possesses the diketone of the optionally oxidized N-terminal methionine residue react with 3,4-diaminobenzoic acid or the salt thereof in the presence of acetic acid and sodium formate,
(8) a method of manufacturing a peptide or the salt thereof which does not possess the optionally oxidized N-terminal methionine residue characterized by having a peptide or the salt thereof which possesses the diketone of the optionally oxidized N-terminal methionine residue react with 3,4-diaminobenzoic acid or the salt thereof in the presence of acetic acid or sodium formate, or formic acid and sodium formate, or formic acid and sodium acetate,
(9) a method of manufacture described in (8) above wherein the peptide or the salt thereof that possesses the diketone of the optionally oxidized N-terminal methionine residue is a peptide or the salt thereof that is obtained by having a peptide or the salt thereof which possesses optionally oxidized N-terminal methionine residue react with an xcex1-diketone,
(10) a method of manufacture described in (8) above characterized by the acetic acid and sodium formate, or formic acid and sodium formate, or formic acid and sodium acetate, being used as a buffering solution of approximately 0.1 to 8 mol/L with a pH of approximately 2 to 9,
(11) a method of manufacturing a peptide or the salt thereof that does not possess N-terminal methionine residue characterized by having a peptide or the salt thereof which possesses the diketone of the optionally oxidized N-terminal methionine residue react with 3,4-diaminobenzoic acid or the salt thereof in the presence of acetic acid and sodium formate,
(12) a method of manufacturing human growth hormone or the salt thereof which does not possess N-terminal methionine residue characterized by having the human growth hormone or the salt thereof that is manufactured via genetic engineering which possesses optionally oxidized N-terminal methionine residue react with glyoxylic acid or the salt thereof in the presence of cupric sulfate and pyridine, and subsequently with 3,4-diaminobenzoic acid or the salt thereof in the presence of acetic acid and sodium formate, or formic acid and sodium formate, or formic acid and sodium acetate,
(13) the use of (i) acetic acid and sodium formate, or formic acid and sodium formate, or formic acid and sodium acetate, and (ii) 3,4-diaminobenzoic acid or the salt thereof, for the purpose of removing the N-terminal methionine residue from a peptide or the salt thereof which possesses optionally oxidized N-terminal methionine residue,
(14) the use of (i) acetic acid and sodium formate, or formic acid and sodium formate, or formic acid and sodium acetate, and (ii) 3,4-diaminobenzoic acid or the salt thereof, for the purpose of removing the diketone of the methionine residue from a peptide or the salt thereof which possesses the diketone of the optionally oxidized N-terminal methionine residue,
(15) the use of (i) acetic acid and sodium formate, or formic acid and sodium formate, or formic acid and sodium acetate, and (ii) 3,4-diaminobenzoic acid or the salt thereof, for the purpose of manufacturing a peptide or the salt thereof which does not possess optionally oxidized N-terminal methionine residue from a peptide or the salt thereof which possesses optionally oxidized N-terminal methionine residue,
(16) the use of (i) acetic acid and sodium formate, or formic acid and sodium formate, or formic acid and sodium acetate, and (ii) 3,4-diaminobenzoic acid or the salt thereof, for the purpose of manufacturing a peptide or the salt thereof which does not possess the diketone of the optionally oxidized N-terminal methionine residue from a peptide or the salt thereof which possesses the diketone of the optionally oxidized N-terminal methionine residue.